CNS progenitor cells may become important therapies for brain and spinal cord injury, degenerative, or genetic disease. To achieve these goals, the fundamental mechanisms by which CNS progenitors choose their cell fate, differentiate, survive, and divide will need to be unraveled. We have previously performed a genetic screen, using a combination of representational difference analysis (RDA), custom, subtracted cDNA microarray screening and in situ hybridization to establish that a large number of known and novel genes are enriched in CNS progenitors, as compared to more differentiated cells. It is our hypothesis that multipotent CNS stem cells will express a set of genes that is responsible for maintaining them in an undifferentiated, proliferative state. In order to test this hypothesis, we will use a combination of approaches to prioritize genes for further study and then we will use functional assays to determine their roles in CNS stem cell biology. We predict that those genes that are shared by multiple stem cell types will be important for the regulation of stem cell proliferation and multipotentiality. First, we will use our custom, subtracted microarray to determine a set of genes that are enriched in neurosphere cultures derived under a variety of conditions, including: variation of trophic factor used to induce proliferation; age of animal at time of culture; and region from which the cultures are derived. We will then determine which subset of these genes is also expressed by hematopoietic stem cells, which we have shown to express numerous genes that are also highly enriched in CNS progenitors. Next, we will use in situ hybridization to identify which of these genes are selectively expressed in progenitor cells in vivo. We will then use functional assays to test our hypothesis that these genes regulate CNS progenitor cell proliferation and/or their potential for differentiation. For those genes that have been successfully "knocked out", we will analyze brains of null mutants and the capacity of progenitors from these animals to divide and differentiate. For other genes, we will use clonal analysis to determine the ability of neurospheres to divide and differentiate in the presence of antisense oligonucleotides - to knock down expression - or after induction of overexpression using lentiviral expression vectors.